The invention relates generally to transcriptional control elements that are tissue specific. More particularly, the invention relates to nucleic acids that contain tissue-specific transcriptional control elements. The invention also relates to methods of using these control elements including for the identification modulators of tissue-specific protein expression.
The transcription factor Pax-6 is known to have a critical role in development of the eye in a number of species [Quiring et al., Science 265:785-789 (1994)]. In Drosophila, homozygous mutation of the Pax-6 orthologue eyeless results in missing eye structures. In humans, heterozygous Pax-6 mutations give the ocular defects aniridia (iris hypolasia [Jordan et al., Nat. Genet. 1:328-332 (1992)]) and Peters"" anomaly (a combination of iris hypolasia and lens-corneal epithelium attachment [Hanson et al., Nature Genetics, 6:168-173 (1994)]). Analogous defects are seen in the Small eye mice, also heterozygous for a Pax-6 mutation [Hill et al., Nature 354:522-525 (1991)]. Homozygous mutation in mice is lethal and results in severe cranio-facial abnormalities including absence of eyes [Hill et al., Nature 354:522-525 (1991)].
Gain-of-function experiments also demonstrate a central role for Pax-6 in development of the eye. In Xenopus, expression of the transcription factor Pax-6 directs induction of the lens [Altmann et al., Dev. Biol. 185:119-123 (1997); U.S. application Ser. No. 08/846,463 filed May 1, 1997 hereby incorporated by reference in its entirety]. Analogously, overexpression of Drosophila Pax-6 in imaginal discs results in the development of ectopic compound eyes [Gehring et al., Genes to Cells 1: 11-15 (1996)]. Remarkably, this activity can be mimicked by Pax-6 from a number of species including, squid [Tomarev et al., Proc. Natl. Acad. Sci. U.S.A. 94:2421-2426 (1997)] and ascidian [Glardon et al., Development 124:817-825 (1997)] arguing that Pax-6 function has been conserved across more than 40 million years of evolution [Gehring et al., Genes to Cells 1:11-15 (1996)].
In animal cap experiments, Pax-6 can induce the expression of the lens-specific marker xcex2B1-crystallin without inducing the general neural marker NCAM. Ectopic Pax-6 expression also results in the formation of ectopic lenses in whole embryos indicating that in vertebrates, as well as Drosophila [Halder, et al., Science, 267:1788-1792 (1995)], Pax-6 can direct the development of major components of the eye. According to NCAM staining, ectopic lenses formed in the whole embryo are only sometimes associated with neural tissue. Furthermore, treatments giving rise to anterior neural tissue result in the expression of both xcex2B 1-crystallin and Pax-6.
In the mouse, Pax-6 has a complex pattern of expression that includes multiple components of the developing eye, as well as neural tube, forebrain neuroepithelium, olfactory epithelium and bulbs, pancreas and pituitary [Grindley et al., Development 121:1433-1422 (1995); Walther et al., Development 113:1435-1449 (1991)]. In the eye, Pax-6 is expressed in the presumptive and mature lens, retina and corneal epithelium [Grindley et al., Development 121:1433-1442 (1995); Walther et al., Development 113:1435-1449 (1991)]. This expression pattern is conserved in different vertebrate species including in Xenopus [Li et al., Development, 124:603-615 (1997)] and chick [Li et al., Dev. Biol., 162:181-194 (1994)].
Although it would be valuable to be able to express selected proteins solely in the lens and/or corneal epithelium, heretofore no lens and/or corneal epithelium specific transcriptional control element has been identified. Therefore, there is a need to identify such a lens and/or corneal epithelium transcriptional control element. Furthermore, there is a need to identify agents that can modulate lens and/or corneal epithelium development including naturally occurring transcription factors.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides a lens transcription control element that can be used to identify factors, both naturally occurring and synthetic, that will modulate lens and/or corneal epithelium development and function. This lens transcription control element can be used to direct transcription in the lens and/or corneal epithelium
The present invention therefore provides a recombinant nucleic acid comprising a lens and corneal epithelium specific transcriptional control element (LCE) that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:1 or a functional derivative thereof. In another embodiment the recombinant nucleic acid contains a LCE that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:3. In a preferred embodiment of this type the hybridization is performed under standard conditions. In a more preferred embodiment the hybridization is performed under stringent conditions.
The present invention also provides a recombinant nucleic acid comprising a lens and corneal epithelium specific transcriptional control element (LCE) that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:2 or a functional derivative thereof. In another embodiment the recombinant nucleic acid contains a LCE that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:4. In a preferred embodiment of this type the hybridization is performed under standard conditions. In a more preferred embodiment the hybridization is performed under stringent conditions.
In one embodiment, the lens specific transcriptional control element contains between 100 to 800 nucleotides. In a preferred embodiment the LCE contains 200 to 600 nucleotides. In a more preferred embodiment the LCE contains 250 to 450 nucleotides. In a particular embodiment, the LCE contains 341 nucleotides. In a preferred embodiment of this type, the LCE has a nucleotide sequence of SEQ ID NO:3. In another embodiment, the LCE has a nucleotide sequence of SEQ ID NO:4.
The LCE of the present invention is preferably a vertebrate control element. In one such embodiment the LCE is a xenopus control element. In an alternative embodiment the LCE is a mammalian control element. In one such embodiment, the mammalian control element is a murine control element. In a preferred embodiment of this type the LCE is a Pax-6 conserved element (PACE) having the nucleotide sequence of SEQ ID NO:1. In an alternative embodiment the PACE has the nucleotide sequence of SEQ ID NO:3. In another such embodiment the mammalian control element is a human control element. In a preferred embodiment of this type the LCE is a Pax-6 conserved element (PACE) having the nucleotide sequence of SEQ ID NO:2. In an alternative embodiment the PACE has the nucleotide sequence of SEQ ID NO:4.
The present invention also includes nucleotide probes that hybridize with a nucleic acid having the nucleotide sequence of SEQ ID NO:1. In a related embodiment the present invention provides a nucleotide probe that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:2. In yet another embodiment the nucleotide probe hybridizes with the nucleotide sequence of SEQ ID NO:3. In still another embodiment the nucleotide probe hybridizes with the nucleotide sequence of SEQ ID NO:4. In a preferred embodiment the hybridization is done under standard hybridization conditions. In a more preferred embodiment the hybridization is performed under stringent conditions.
The present invention further provides a recombinant DNA molecule that comprises a recombinant nucleic acid that contains an LCE of the present invention and a coding sequence operatively linked to the recombinant nucleic acid. In a preferred embodiment of this type the LCE has the nucleotide sequence of SEQ ID NO:1. In a related embodiment, the LCE has the nucleotide sequence of SEQ ID NO:2. In still another embodiment the LCE has the nucleotide sequence of SEQ ID NO:3. In yet another embodiment the LCE has the nucleotide sequence of SEQ ID NO:4.
The recombinant DNA molecules of the present invention can further comprise a promoter or a minimal promoter that functions in conjunction with the LCE of the recombinant nucleic acid. In a preferred embodiment of this type, the LCE is adjacent to the promoter or minimal promoter. The present invention further provides expression vectors which comprise the recombinant DNA molecules of the present invention. The coding sequences contained in the expression vectors can encode a marker protein or a therapeutic protein. In one embodiment the marker protein is green fluorescent protein. In another embodiment the marker protein is xcex2-galactosidase.
The present invention also provides transgenic animals which comprise cells containing an expression vector of the present invention. In a preferred embodiment the transgenic anal is a mammal. In a more preferred embodiment the mammal is a mouse. Such expression vectors comprise a coding sequence that is operatively linked to a promoter or a minimal promoter and under the control of an LCE or PACE of the present invention.
Another aspect of the present invention provides methods of expressing the coding sequence contained in the expression vectors of the present invention in a cell.
The present invention further provides a method of directing the expression of the coding sequence contained within an expression vector of the present invention to the presumptive lens ectoderm while excluding expression in neural components of the eye. In one such embodiment the expression vector is placed into an animal zygote. In a preferred embodiment of this type placing of the expression vector into the animal zygote is performed by injection. In a particular embodiment the animal zygote is a xenopus zygote. In another such embodiment the animal zygote is a mammalian zygote. In a preferred embodiment of this type the mammalian zygote is a murine zygote.
In addition, the present invention provides a method of detecting the lens and/or corneal epithelium specific transcription of an mRNA encoding a marker protein in an animal zygote. In one such embodiment, an expression vector of the present invention is placed into an animal zygote. The expression vector contains a coding sequence that is operatively linked to a promoter or a minimal promoter and under the control of an LCE of the present invention. The coding sequence encodes a marker protein. The zygote is allowed to develop, and an mRNA encoding the marker protein is transcribed. The transcription of the mRNA encoding the marker protein is then detected. In a related embodiment, the mRNA is expressed and the step of detecting the transcription of the mRNA encoding the marker protein is performed by detecting the marker protein.
The present invention further provides a method of identifying an agent or drug that modulates lens and/or corneal epithelium development. In one such embodiment, an expression vector of the present invention is placed into an animal zygote in the presence of the potential drug or agent. The expression vector contains a coding sequence operatively linked to a promoter or a minimal promoter and under the control of an LCE of the present invention. In a particular embodiment the minimal promoter is from the hsp68 gene. The coding sequence encodes a marker protein.
The zygote is allowed to develop under conditions in which in the absence of the potential agent or drug, an mRNA encoding the marker protein is transcribed. The amount of transcription of the mRNA encoding the marker protein is determined. A potential agent or potential drug is identified as an agent or drug when the amount of transcription of the mRNA encoding the marker protein in the presence of the potential agent is different than in its absence. An agent or drug so identified is further identified as an agonist if the amount of transcription of the mRNA encoding the marker protein in the presence of the agent or drug is greater than in its absence. Analogously, the agent or drug is further identified as an antagonist when the amount of transcription of the mRNA encoding the marker protein in the presence of the potential drug or agent is less than in its absence. In another particular embodiment the agent that is identified, is a transcription factor. In yet another embodiment, the mRNA that is transcribed is also expressed and the step of determinig the amount of transcription of mRNA encoding the marker protein is performed by determining the amount of the marker protein.
The present invention further provides a method of identifying a transcription factor that modulates lens and/or corneal epithelium development. In one such embodiment, an expression vector of the present invention is placed into an animal zygote in the presence of a potential transcription factor. Such an expression vector comprises a coding sequence that is operatively linked to a promoter or preferably a minimal promoter and under the control of an LCE or PACE of the present invention. In one particular embodiment the minimal promoter is from the hsp68 gene.
The zygote is allowed to develop under conditions where the transcription of an mRNA encoding the marker protein requires the presence of the transcription factor. The transcription of the mRNA encoding the marker protein is then detected. A potential transcription factor is identified as a transcription factor that modulates lens and/or corneal epithelium development when the transcription of the mRNA encoding the marker protein is detected in the presence of the potential transcription factor. In a related embodiment, the mRNA that is transcribed is also expressed and the step of detecting the transcription of the mRNA encoding the marker protein is performed by detecting the marker protein.
The present invention further includes a method of identifying a transcription factor that modulates a gene which comprises an LCE or PACE of the present invention. In one such embodiment an expression vector of the present invention is placed into a cell in the presence of a potential transcription factor. The expression vector comprises a coding sequence encoding a marker protein that is operatively linked to a promoter or a minimal promoter and under the control of the LCE. In a particular embodiment of this type, the minimal promoter is from the hsp68 gene. The cell is cultured in an appropriate cell culture medium under conditions that provide for transcription of an mRNA encoding the marker protein by the cell, but requires the presence of the transcription factor. The transcription of the mRNA encoding the marker protein is detected and a potential transcription factor is identified as a transcription factor that modulates the gene when the transcription of the mRNA encoding the marker protein is detected. In a related embodiment the mRNA that is transcribed is also expressed, and the step of detecting the transcription of the mRNA encoding the marker protein is performed by detecting the marker protein.
The present invention further provides a method of identifying a binding partner for an LCE or PACE of the present invention. In one such embodiment, a candidate binding partner is contacted with a recombinant nucleic acid which comprises an LCE or PACE of the present invention. The binding of the candidate binding partner with the recombinant nucleic acid is detected, and a candidate binding partner is identified as a binding partner when the candidate binding partner is detected as binding to the recombinant nucleic acid. In one such embodiment the LCE has the nucleotide sequence of SEQ ID NO:1. In another such embodiment the LCE has the nucleotide sequence of SEQ ID NO:2. In still another embodiment the LCE has the nucleotide sequence of SEQ ID NO:3. In yet another such embodiment the LCE has the nucleotide sequence of SEQ ID NO:4.
In a related embodiment, the method of identifying a binding partner for an LCE or PACE of the present invention comprises placing a recombinant nucleic acid comprising an LCE or PACE of the present invention on a solid support. The solid support is contacted with a candidate binding partner under conditions in which a binding partner can bind to the recombinant nucleic acid. A solid support is washed and candidate binding partners that do not bind the recombinant nucleic acid are removed. The candidate binding partner bound to the solid support is detected. A candidate binding partner is identified as a binding partner if it binds to the solid support. In a preferred embodiment of this type, the candidate binding partner is a potential transcription factor for a gene that comprises the LCE or PACE. In this case, the binding partner that is identified is a transcription factor for the gene which comprises the LCE or PACE. In one such embodiment, the solid support is a nitrocellulose filter. In one such embodiment the LCE has the nucleotide sequence of SEQ ID NO:1. In another such embodiment the LCE has the nucleotide sequence of SEQ ID NO:2. In still another embodiment the LCE has the nucleotide sequence of SEQ ID NO:3. In yet another such embodiment the LCE has the nucleotide sequence of SEQ ID NO:4.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.